Project Summary The drug therapies against the central nervous system (CNS) disorders, such as brain cancer, stroke and Alzheimer's disease, are very limited because of the challenges associated with delivering drugs across the blood-brain barrier (BBB) into the brain. There is a need to develop novel strategies to enhance the delivery of CNS drugs. We have identified a peptide, called the CL peptide, that demonstrates an ability to deliver a small-molecule cargo across a model endothelial barrier. However, little is known about the biophysical properties of this peptide that give it the barrier-penetrating property. In this project, we propose to investigate the biophysical parameters for the peptide-based barrier penetration by working at the intersection of peptide engineering, membrane biophysics, in vitro BBB model development, and microfluidic device design. In Aim 1, we propose to investigate the sequence and secondary structure requirements for the high transcellular transport with the long-term goal of developing design rules for the barrier-penetrating peptides. We will synthesize variants of the CL peptide to test the effect of peptide's charge, hydrophobicity, and amphipathicity on transcellular transport rate. In Aim 2, we propose to use a microfluidic device and confocal microscopy to measure the individual rates of the peptide variants' entry and exit out of cells. We will test the hypothesis that the barrier-penetrating properties are associated with higher exit rates, when compared to other peptides with low transcellular transport rates.